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Notebook
You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.

Lab Protocols

Competent Cell with Calcium Chloride

1. Materials

• Solid LB medium - 1 plate
• Liquid LB medium
• 2 250ml centrifuge tubes
• Centrifuge
• Cell sample (DH5-alpha in our case)
• Autoclave
• Inoculation loop
• Liquid nitrogen
• 37°C oven
• CaCl₂:
• 60mM CaCl₂
• 10mM HEPES
• 15% glicerol
• H₂O to 100ml

2. Methods

Day 1

1. Risk the LB plate with the cell sample (DH5-alpha)
2. Incubate the plate at 37°C overnight

Day 2

1. Sterilize in the Autoclave
• 100ml of liquid LB medium
• 100 ml of CaCl₂
• 2 250ml centrifuge tubes
2. Prepare a 6ml LB inoculum with a DH5-alpha colony and incubate at 37°C/250rpm overnight

Day 3

1. Add 2ml of the inoculum in 100ml of liquid LB medium. Incubate at 37°C/250rpm until the OD reaches 0,375
2. Distribute the volume in 2 250ml centrifuge tubes (pre chilled) and spin at 10000rpm/7min/4°C
3. Discard the supernatant and ressuspend the pellet in 10ml of CaCl₂ solution
4. Spin the tubes at 7000rpm/7min/4°C
5. Discard the supernatant and ressuspend the pellet in 10ml of CaCl₂ solution. Incubate 30min on ice
6. Spin at 7000rpm/7min/4°C
7. Discard the supernatant and ressuspend the pellet in 2ml of the CaCl₂ solution
8. Distribute the 2ml in 500ul tubes adding 50ul in each.
9. Freeze the samples in liquid nitrogen
10. Stock at -80°C

Transformation in Escherichia coli (DH5-alpha)

1. Materials

• 1,5ml tube
• Styrofoam box with ice
• Agar plate with antibiotic
• Competent cells
• Plasmidial DNA
• Centrifuge
• Water bath at 42°C
• Liquid LB medium
• Shaker

2. Methods

1. Briefly spin the competent cells and put then on ice
2. Add 50ng of plasmidial DNA in a 1,5ml tube
3. Add the 50ul of competent cell in the same tube
4. Keep the tube on ice for 25min
5. Put the tube in a 42° water bath for 2 min
6. Put the tube on ice for 5 min
7. Add 200ul of liquid LB
8. Incubate at 250rpm/37°C/1 hour
9. Plate the the solution in a Agar plate with the appropriate antibiotic
10. Incubate the plate at 37°C overnight

Assemblies

• Assembly Form
• AI
• AII
• AIII
• AV
• AVI
• BII
• BIII
CII
• DI
• KI
• KIII
• KIV
• KVI
• KVIII
• KX
• KXI

Life Inside the LAB

July

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
30/06 PCR lasR (BBa_C0079) for amplification + RBS addition using primers	01/07 Purification of lasR PCR product	02/07 Transformation of the Biobricks: BBa_143012 BBa_K081001 BBa_E0840 Inoculum of the Biobricks sent by iGEM HQ: BBa_K316037 BBa_K316018 BBa_K316015	03/07 Transformation of the Biobricks sent by the Imperial College Team: BBa_K316016 BBa_K143031 Miniprep, Quantification of the plasmidial DNA samples and Restriction Analysis: BBa_143012 BBa_K081001 BBa_E0840	04/07	05/07	06/07 Inoculum: BBa_143012 BBa_K081001 BBa_E0840
07/07 Miniprep, Quantification and Restriction Analysis: BBa_143012 BBa_K081001 BBa_E0840 Transformation: BBa_B0015	08/07	09/07 Inoculum: BBa_B0015	10/07 Miniprep, Quantification and Restriction Analysis: BBa_B0015 (restriction analysis fail, repeat) PCR qteE for amplification + RBS addition using primers Ligation of RBS+lasR in PTZ57R/T vector (instaclone kit)	11/07 Prepare 40% glycerol solution Transformation: RBS + lasR (BBa_C0079) Purification: RBS + qteE	12/07	13/07
14/07 Assembly:AIII Digestion EXSP Prepare X-gal Prepare more LB medium (solid + liquid)	15/07 Assembly:AIII Quantification of digestion EXSP products Replate RBS+lasR Ligation of PCR qteE in pGEM T-easy vector Transformation: BBa_J04450 Restriction analysis: BBa_B0015	16/07 Tranformation: BBa_K823003 RBS+qteE Electrophoresis analysis: BBa_B0015	17/07 Glycerol Stock and Miniprep: BBa_J04450 RBS+lasR BBa_B0015 Digestion: BBa_J04450 (EP) BBa_J04450 (XS) Inoculum: BBa_K823003 Transformation BBa_K143055	18/07 Glycerol Stock and Miniprep: BBa_J04450 BBa_B0015 Gel Purification: BBa_J04450 (EP) BBa_J04450 (XS) Restriction analysis: BBa_K823003	19/07	20/07
21/07 Transformation: BBa_316016 Inoculum: BBa_K143055 RBS+qteE Assembly KI: Digestion EXSP	22/07 Inoculum: BBa_K316016 Miniprep, Restriction analysis and Digestion: RBS+qteE Assemblies AIII and KI: Gel Purification Ligation	23/07 Assemblies AIII and KI: Transformation Prepare more competent cells Prepare LB medium (solid and liquid) Inoculum BBa_K316016 Prepare RBS+lasR and RBS+qteE for sequencing	24/07 Assemblies AIII and KI: Inoculum Miniprep: BBa_K316016	25/07 Assemblies AIII and KI: Glycerol Stock Miniprep Restriction analysis	26/07	27/07
28/07 Assembly AIII: Restriction analysis (+NdeI enzyme) OBS: This analysis was reapeted because we had problems confirming the assembly. The issue was that bot the construction and the pSB1C3 vector had similar size. Check out the Lab Journal report of the day for more information. Assembly AII, AV and AVI: Digestion EXSP	29/07 >Assembly AII, AV and AVI: Gel Purification Ligation	30/07 >Assembly AII, AV and AVI: Transformation	31/07 Assembly AII, AV and AVI: Inoculum PCR BBa_K143055 for RBS removal OBS: This was one of the parts sent by the Imperial College Team. Because of a comunication failure we assumed that this part was comming without the RBS, so we included a RBS in the parts we were going to use with this Lac promotor :) PCR of Plac BBa_K143055 was followed by a Gel Purification and Ligation in the PUC19 vector

August

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
				01/08 Transformation: BBa_K143055 Assembly AII, AV and AVI: Glycerol Stock Miniprep Restriction analysis	02/08	03/08
04/08 Inoculum E. coli DH5-alpha KI Plac BBa_K143055 Transformation BBa_P0312 Assembly AV: Prepare more inoculum Repeat Restriction analysis (for more info go to the day's report)	05//08 Prepare assemblies KI for cytometry characterization Miniprep: Plac BBa_K143055 Inoculum BBa_P0312 Assembly AV: Miniprep And once again! Restriction analysis (this BBa_K316016 is tough)	06/08	07/08 MIniprep and Restriction analysis: BBa_P0312	08/08	09/08	10/08
11/08 Prepare Plac BBa_K143055 for sequencing Analyse the RBS+qteE and RBS+lasR sequencing file	12/08 Digestion: RBS+qteE RBS+lasR OBS: this digestion was performed so we clone this parts in the pSB1C3 vector to put them in Biobrick standard	13/08 Assemblies BII and BIII: Digestion EXSP	14/08 Gel Purification and Ligation in pSB1C3 for BBiobrick standard: RBS+qteE RBS+lasR Assemblies BII and BIII: Gel Purification OBS: we had some problems here with the purification and we lost all our digestions, so we're repeating the digestion	15/08	16/08 Assemblies BII and BIII: Digestion EXSP	17/08 Assemblies BII and BIII: Gel Purification
18/08 Assemblies BII and BIII: Ligation Prepare LB medium (liquid and solid)	19/08 Assemblies BII and BIII: Transformation	20/08 RBS+qteE, RBS+lasR and Assemblies BII and BIII: Inoculum	21/08 RBS+qteE, RBS+lasR and Assemblies BII and BIII: Glycerol Stock Miniprep Restriction analysis	22/08 Prepare LB medium (liquid and solid) Assembly BII: Repeat restriction analysis adding the enzyme NdeI (for more info check out the Lab Journal)	23/08	24/08
25/08 Inoculum BBa_J04450 BBa_K143055 PCR BBa_K143055 with higher melting temperature (check out our Lab Journal for more info)	26/08 Assemblies AI, CII, KIII, KIV and KVI Digestion EXSP	27/08 Assemblies AI, CII, KIII, KIV and KVI Gel Purification	28/08	29/08	30/08	31/08

September

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
01/09 Assembly CII Digestion of the part BII with EcoRV (for more info check out our Lab Journal) Assemblies AI, CII, KIII, KIV and KVI: Ligation	02/09 Assemblies AI, CII, KIII, KIV and KVI: Transformation	03/09 Assemblies AI, CII, KIII, KIV and KVI: Inoculum	04/09 Assemblies AI, CII, KIII, KIV and KVI: Glycerol Stock Miniprep Restriction analysis	05/09	06/09	07/09
08/09 QuickChange PCR to mutate the cleavage site of the BBa_316037 to a Cathepsin S recognition site Transformation of the QuickCHange PCR product	09/09	10/09 The QuickChange protocol didn't work well. So try again with more attention! QuickChange PCR to mutate the cleavage site of the BBa_316037 to a Cathepsin S recognition site Transformation of the QuickCHange PCR product Assemblies DI, KVIII, KX and KXI: Digestion EXSP	11/09 Prepare inoculum of the mutated BBa_K316037	12/09	13/09	14/09
15/09	16/09	17/09	18/09	19/09	20/09	21/09
22/09	23/09	24/09	25/09	26/09	27/09	28/09
29/09	30/09

October

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
	30/07	01/08	02/08	03/08	Points	Points
First Name	Last Name	Points	Points	Points	Points	Points
First Name	Last Name	Points	Points	Points	Points	Points
First Name	Last Name	Points	Points	Points	Points	Points
First Name	Last Name	Points	Points	Points	Points	Points

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